MICROBIAL AND ENZYMATIC SYNTHESIS OF SELENIUM-CONTAINING POLYPEPTIDES AND THEIR ACTION
| Project/Area Number |
63560104
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
発酵・醸造
|
|---|
| Research Institution | OKAYAMA UNIVERSITY |
|---|
Principal Investigator |
TANAKA Hidehiko OKAYAMA UNIV., FACULTY OF AGRICULTURE, PROFESSOR, 農学部, 教授 (90065912)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
INAGAKI Kenji OKAYAMA UNIV., THE GRADUATE SCHOOL OF NATURAL SCIENCE AND TECHNOLOGY, INSTRUCTOR, 大学院自然科学研究科, 助手 (80184711)
ESAKI Nobuyoshi KYOTO UNIV., INSTITUTE FOR CHEMICAL RESEARCH, ASS. PROF., 化学研究所, 助教授 (50135597)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Keywords | SELENOMETALLOTHIONEIN / SELENIUM BIOCHEMISTRY / SUPPRESSION OF NONSENCE CODON / GENE SYNTHESIS / SELENOGLUTATHIONE / DETOXIFICATION OF HEAVY METALS / 遺伝子合成 / 遺伝子発現 / ナンセンスコドンの抑制 |
|---|
| Research Abstract |
A selenocysteine residue occurs as an integral Moiety in the active center of the selenium-containing enzymes such as glycine reductase, formate dehydrogenase and glutathione peroxidase. The catalytic role of the seleno- cysteine residue is attributed to high reactivity of the selenol group of the selenocysteine residues: selenols have much lower redox potential than the sulfur counterparts. Thus, attention has been paid to synthesis of sele- nocysteine-containing polypeptides and proteins which may have unique properties that the sulfur counterparts do not have. The present study was performed to synthesize the novel selenocysteine-containing peptides, seleno- metallothionein and selenoglutathione, and to elucidate their structures and function.
1. Metallothioneins (MTs) are a class of low-molecular weight and cysteine rich proteins, which bind with high amount of metal ions such as Zn, Cd and Cu. Neuros ora Cu-MT consists of only 25 amino acids and binds 6 Cu ions to 7 cysteinyl resid
… More
ues: the smallest MT thus far isolated. We synthesized selenium analogue of the MT peptide (selenometallothionein (SeMT)), in which all the cysteine residues are replaced by selenium counterparts, to study the structure of the Cu-selenoate cluster in a SeMT-Cu complex. In contrast to the native Cu-MT complex, 3 Cu ions are coordinated in the form of a single Cu(II)-selenoate cluster. To obtain further insight into the structure of the complex, we have attempted EXAFS analysis. The XANES oscillations of the Cu complexes of MT and SEMT revealed a remarkable similarity. This indicates that the structure of CU(II) thiolate cluster in the CuMT is similar to that of Cu(II)-selenoate cluster in the selenium counterpart.
2. The gene coding. for the Neurospora MT was synthesized and fused with the gene of beta-gal. The MT-beta-gal fused gene was expressed in E. coli to produce a fused protein. The spectral features of the MT prepared with the fused protein were similar to those of the corresponding native MTs. TGT encoding the first cysteine residue near the amino terminal in the MT-beta-gal fused gene was replaced by TGA. The mutant gene was constructed by a simple method newly developed. The method is based on the use of denatured double-stranded DNA as a template and phosphorothioate-modified nucleotide in the elongation reaction. beta-Gal activity was detected in the transformed when cultivated anaerobic only, and selenium was incorporated into the fused protein. These results suggest that the TGA suppression factors formed inducibly under anaerobic conditions regulate SeCys incorporation into proteins, and that these factors introduce SeCys not only into inherent SeCys-containing proteins whose SeCys is encoded by the TGA codon, but also into other proteins that have no SeCys by introduction of TGA into the gene artificially.
3. Selenoglutathione, a selenium analogue of glutathione was synthesized with an Applied Biosystems peptide synthesizer 430A. The compound synthesized was oxidized form. Less
|
Report
(3 results)
Research Products
(17 results)
